Thanks to Diana Icaza-Culaki, Cristal Sampson and Theologia Karagiorgis for their hard work on this project.

These experimental protocols have been approved by the Institutional Animal Care and Use Committee certifying that all subjects and procedures are in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
1. Subjects
<ol>
	<li>Purchase and/or breed male Sprague-Dawley rats (260 - 300 g).</li>
	<li>House rats individually in wire mesh cages. Maintain them on a 12:12 hr light/dark cycle with rat chow and water available ad libitum.</li>
	<li>Assign appropriate samp...

The goal of the study was to determine if the source (VTA) and forebrain projection targets (NAC, AMY, mPFC) of DA reward-related neurons were simultaneously activated after novel ingestion of fat and sugar in rats using the cellular c-fos technique. The present study is a detailed description of the protocols of a study published previously69. It was hypothesized that the VTA, its major projection zones to the prelimbic and infralimbic mPFC, the core and shell of the NAC and the basolateral and central-cortic...

Brain dopamine (DA) has been implicated in central responses to intake of palatable sugars through proposed hedonic1,2, effort-related3 and habit-based4,5 mechanisms of action. The primary DA pathway implicated in these effects originates in the ventral tegmental area (VTA), and projects to the nucleus accumbens (NAC) core and shell, the basolateral and central-cortico-medial amygdala (AMY), and the prelimbic and infralimbic medial prefrontal cortex (mPFC) (see reviews6,7). The VTA has been implicated in sucrose intake8,9, and DA release is observed following sugar intake in the NAC10-15, AMY16,17 and mPFC18-20. Fat intake also stimulates DA NAC release21, and another DA-rich projection zone to the dorsal striatum (caudate-putamen) has been also associated with DA-mediated feeding22,23. Kelley24-27 proposed that these multiple projection zones of this DA-mediated system formed an integrated and interactive distributed brain network through extensive and intimate interconnections28-34.
In addition to the ability of DA D1 and D2 receptor antagonists to reduce intake of sugars35-37 and fats38-40, DA signaling has also been implicated in mediating the ability of sugars and fats to produce conditioned flavor preferences (CFP)41-46. Microinjections of a DA D1 receptor antagonist into the NAC, AMY or mPFC47-49 eliminate acquisition of CFP elicited by intragastric glucose. Whereas microinjections of either DA D1 or D2 receptor antagonists into the mPFC eliminates acquisition of fructose-CFP50, the acquisition and expression of fructose-CFP are differentially blocked by DA antagonists in the NAC and AMY51,52.
The c-fos technique53,54 has been employed to investigate neural activation induced by palatable intake and neural activation. The term &#34;c-fos activation&#34; will be used throughout the manuscript, and is operationally defined by increased transcription of c-Fos during neuronal depolarization. Sucrose intake increased fos-like immunoreactivity (FLI) in the central AMY nucleus, the VTA as well as the shell, but not core, of the NAC55-57. Whereas sucrose intake in sham-feeding rats significantly increased FLI in the AMY and the NAC, but not the VTA58, intragastric sucrose or glucose infusions significantly increased FLI in the NAC and central and basolateral nuclei of the AMY59,60. Repeated addition of sucrose to scheduled chow access increased FLI in the mPFC as well as the NAC shell and core61. A sucrose concentration downshift paradigm revealed that the greatest FLI increases occurred in the basolateral AMY and NAC, but not the VTA62. Following conditioning, extinction of sugar-related natural reward behaviors increased FLI in the basolateral AMY and the NAC63. Moreover, pairing sugar availability to a tone resulted in the tone subsequently increasing FLI levels in the basolateral AMY64. High-fat intake also increased FLI in NAC and mPFC sites65-67.
Most of the previously cited studies examined sugar and fat effects on c-fos activation in single sites that do not provide information about identification of reward-related distributed brain networks24-27. Further, many of the studies also did not delineate the relative contributions of sub-areas of the NAC (core and shell), AMY (basolateral and central-cortico-medial) and mPFC (prelimbic and infralimbic) that could potentially be examined by the advantage of excellent spatial, single-cell resolution in c-Fos mapping68. Our laboratory69 recently used c-fos activation and simultaneously measured alterations in the VTA DA pathway and its projection zones (NAC, AMY and mPFC) after novel ingestion of fats and sugars in rats. The present study describes the procedural and methodological steps to simultaneously analyze whether acute exposure to six different solutions (corn oil, glucose, fructose, saccharin, water and a fat emulsion control) would differentially activate FLI in sub-areas of the NAC, AMY, mPFC as well as the dorsal striatum. This simultaneous detection of differences allowed confirmation of significant effects on FLI in each site and determination as to whether changes in one particular site correlated with changes in related sites, thereby providing support for a distributed brain network24-27. These procedures tested whether the VTA, the prelimbic and infralimbic mPFC, the core and shell of the NAC, and the basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of glucose (8%), fructose (8%), corn oil (3.5%) and saccharin (0.2%) solutions.

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			<td height="21" style="height:21px;">Nalgene Centrifuge Tubes</td>
			<td>Lab Products, Seaford, DE</td>
			<td>10-0501</td>
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			<td>F0127</td>
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			<td>Sigma Chemical Co</td>
			<td>G8270</td>
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			<td>MBF Bioscience</td>
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			<td>12-550-343</td>
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			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
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			<td>Loew-Cornell&#160;</td>
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			<td>Fisher Scientific</td>
			<td>3527</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
			<td>1395-1L</td>
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			<td>Fisher Scientific</td>
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			<td>World Precision Instruments</td>
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			<td>World Precision Instruments</td>
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			<td>Biologix Research Co.</td>
			<td>10-0501</td>
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			<td>Biologix Research Co.</td>
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			<td>Madell Corporation&#160;&#160;</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
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			<td>Fisher Scientific</td>
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</table>

simultaneous detection of c-fos activation from mesolimbic and mesocortical dopamine reward sites following naive sugar and fat ingestion in rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

All representative results described below have been published previously69, and are re-presented here to support &#34;proof of concept&#34; in indicating the effectiveness of the technique.
Solution Intakes 
Significant differences in baseline saccharin intakes were observed over the first four days for all animals (F(3,108) = 57.27, p &#60; 0.001) with intakes (Day 1: 1.3 (&#177; 0.2) ml; Day 2...

Watch this Scientific Journal Video about Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats at JoVE.com

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

The overall goal of this experimental protocol is to determine whether the VTA, and it's major mesotelencephatic DA projection zones would display coordinate and simultaneous c-Fos activation after novel intake of corn oil, glucose, fructose, or saccharine solutions. This method can help answer key questions in the neurobiology field, such as motivation, and identify whether multiple brain sites are activated in a coordinated fashion in response to palatable stimuli. The main advantage of this technique is that it combines the established behavioral assays, cellular activation techniques, and the statistical analyses.To set up the apparatus, use a calibrated centrifuge tube with a robber stopper, and a 45-degree angle metal sipper tube to provide an accurate measurement of the solution presented to the rat. Secure it to the home cage by a taut metal spring, and to allow visibility of the calibration. Three to five days before the training, restrict food rations to the rat to reduce its body weight to 85%of its original weight, in order to increase motivation to consume the solution.In the meantime, provide 10 milliliters of pre-training solution, with a 0.2%saccharine, to the animal for four days, over a one-hour session, to maximize the probability that the rat will sample the subsequent test solution with short latency. Weigh the tube before and after each session to obtain the intake measurement. Then, perform an intake test on the fifth day on subgroups receiving one of the six solutions.Ensure that the rats sample solution with short latency, and exclude the subjects from the study if this requirement is not met. To prepare the tissue, after sacrificing the animal, remove the brain from the skull quickly. Use the rongeurs to carefully crack and remove the bone from the brain, starting from rear to front.Work in the area below the cerebellum, and ensure the rongeurs are between the bone and the meningeal pia mater. Once the top and the sides of the skull are removed, use a small spatula to lift the brain from its base, and snip the cranial nerves with small scissors. Then, fix the brain in 4%paraformaldehyde solution overnight at four degrees Celsius.The next day, place the brain in the sucrose solution at room temperature, until it settles at the bottom of the container. After that, remove the olfactory bulb. Subsequently, remove the cerebellum and the pons.Then, mount the brain coronally, with the caudal part fixed to the stage of the microtome, and cut coronal sections of 40 mirometers. In this procedure, treat the sections with five milliliters of 5%normal goat serum, and 0.2%Triton x-100 in PBS, for one hour. Next, incubate the treated sections with primary antibody at four degrees Celsius for 36 hours, in the wells containing one milliliter of PBS.After 36 hours, rinse the sections three times with five milliliters of PBS for 10 minutes each time. Then, incubate the sections with secondary antibody at room temperature for two hours, in the wells containing one milliliter of PBS. Afterward, rinse the sections three times in five milliliters of PBS for 10 minutes each time.Next, incubate the rinsed sections for two hours in a commercially-available Avidin horseradish peroxidase mixture. After two hours, rinse the sections three times in five milliliters of PBS, for ten minutes each time. Subsequently, react the sections with DAB in the presence of hydrogen peroxide, for five to ten minutes.After that, double label the VTA sections by incubating them with a tyrosine hydroxylase antibody, in five millilters of PBS, overnight at four degrees Celsius. The next day, rinse the sections three times in five milliliters of PBS, for ten minutes each time. Following that, incubate the sections with secondary antibody in five milliliters of PBS, at room temperature, for two hours.After two hours, rinse the sections three times in five milliliters of PBS, for ten minutes each time. To visualize the antibody using a secondary antibody peroxidase complex, react it with a combination of DAB, and a 0.3%nickel sulfate solution, for five to ten minutes. Count the cells by double-clicking the software icon.Go to the menu bar, click on Acquisition, and then Live Image. After that, bring the region of interest into focus, and click the screen to establish a reference point. Next, go to the grid toolbar, and click Display Grid, and Use Grid Labels.Outline the region of interest with a predetermined trace. Then, count all the cells in each region of interest, and select Plus in the left-hand sidebar to register counts of the c-Fos cells. Consider a cell positive for c-Fos when a defined dark-red circle is observed.Repeat this process for each site, and ensure the inter-rater reliability of the two uninformed raters for each section in each region of interest, always exceeds 0.8. Afterward, count the c-Fos positive neurons in the regions of interest. Delineate whether c-Fos immunoreactivity is present in the TH-positive and TH-negative cells in the VTA.This image shows that the fat or sugar intake differentially increases c-Fos activation in the entire amygdala, as well as in the basolateral, and central-cortico-medial amygdala subareas. In this image, actual amygdala c-Fos activation was observed in animals exposed to intakes of corn oil, glucose, as well as fructose. C-Fos activation in the VTA was also observed in animals exposed to corn oil and water.Black arrows indicate the representative, double-labeled, TH c-Fos positive cells, while gray arrows indicate the representative c-Fos only cells. Once mastered, this technique can be done in three to four weeks, if performed properly. While attempting this procedure, it's important to remember to follow precise methodology, storage and counting.Thanks for watching, and good luck with your experiments.

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JoVE Journal

Neuroscience

8.9K Views.  CUNY, New York, NY. The overall goal of this experimental protocol is to determine whether the VTA, and it's major mesotelencephatic DA projection zones would display coordinate and simultaneous c-Fos activation after novel intake of corn oil, glucose, fructose, or saccharine solutions. This method can help answer key questions in the neurobiology field, such as motivation, and identify whether multiple brain sites are activated in a coordinated fashion in response to palatable stimuli. The main advantage of thi...

Video: Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats